EP3693742B1

EP3693742B1 - Methods of detecting prostate cancer

Info

Publication number: EP3693742B1
Application number: EP20166589.0A
Authority: EP
Inventors: Harry Stylli; Colleen KELLY
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-09-11
Filing date: 2015-09-11
Publication date: 2022-04-06
Anticipated expiration: 2035-09-11
Also published as: MA40636A; AU2015314813B2; US20170275704A1; EP4075139A1; EP4075139B1; AU2022203356A1; EP3693742A3; EP3191846A1; US10626464B2; EP3693742A2; AU2015314813A1; US20200318201A1; WO2016040843A1; EP3191846A4

Description

Field of the Invention

This invention relates generally to using biological markers for the diagnosis, prognosis, and monitoring of prostate cancer.

Background of the Invention

Early diagnosis of prostate cancer often increases the likelihood of successful treatment or cure of such disease. WO 2009/092068 provides methods and compositions for diagnosing the presence of a cancer cell in an individual. WO 2013/188846 describes methods of using a sample with multiple analytical components in the diagnosis. WO 2013/003384 refers to a set of genes and gene products that can inform about the risk of cancer progression and recurrence, as well as methods of their use. US 2010/0137164 relates to the identification and use of gene expression profiles with clinical relevance to prostate cancer. T. Nakagawa et al. (2008), PLOS one, 3(5) describes a tissue biomarker panel for predicting systemic progression after PSA recurrence postdefinitive prostate cancer therapy. WO 2014/164362 is prior art under Article 54(3) EPC and provides biological markers for the diagnosis, prognosis, and monitoring of prostate cancer. Current diagnostic methods, however, depend largely on population-derived average values obtained from healthy individuals. Personalized diagnostic methods are needed that enable the diagnosis, especially the early diagnosis, of the presence of prostate cancer in individuals who are not known to have the cancer or who have recurrent prostate cancer.
Leukocytes begin as pluripotent hematopoietic stem cells in the bone marrow and develop along either the myeloid lineage (monocytes, macrophages, neutrophils, eosinophils, and basophils) or the lymphoid lineage (T and B lymphocytes and natural killer cells). The major function of the myeloid lineage cells (e.g., neutrophils and macrophages) is the phagocytosis of infectious organisms, live unwanted damaged cells, senescent and dead cells (apoptotic and necrotic), as well as the clearing of cellular debris. Phagocytes from healthy animals do not replicate and are diploid, i.e., have a DNA content of2n. On average, each cell contains <10 ng DNA, <20 ng RNA, and <300 ng of protein.
Non-phagocytic cells arc also diploid and arc not involved in the internalization of dead cells or infectious organisms and have a DNA index of one.
The lifetime of various white blood cell subpopulations varies from a few days (e.g., neutrophils) to several months (e.g., macrophages). Like other cell types, leukocytes age and eventually die. During their aging process, human blood- and tissue-derived phagocytes (e.g., neutrophils) exhibit all the classic markers of programmed cell death (i.e., apoptosis), including caspase activation, pyknotic nuclei, and chromatin fragmentation. These cells also display a number of "eat-me" flags (e.g., phosphatidylserine, sugars) on the extracellular surfaces of their plasma membranes. Consequently, dying and dead cells and subcellular fragments thereof are cleared from tissues and blood by other phagocytic cells.
The prostate specific antigen is currently one of the most widely used diagnostic measures used to detect prostate cancer. However, false negatives and false negatives are common, resulting in mistreatment of patients with no prostate cancer or overtreatment of patients with non-lethal prostate cancer. Thus, improved methods for detecting prostate cancer are needed.

Summary of the Invention

The present invention is defined by the claims.
Hence, the present invention relates to a method for diagnosing or aiding in the diagnosis of prostate cancer in a subject, or for assessing the risk of developing prostate cancer in a subject, or for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the steps of: (a) measuring the levels of ten or more markers selected from the group of markers identified in Table 1 as Signature 1 markers in a population of the subject's macrophage cells or neutrophil cells; (b) measuring the levels of the ten or more selected Signature 1 markers in a population of the subject's non-phagocytic cells; and (c) identifying a difference between the measured levels of the ten or more selected Signature 1 markers in steps (a) and (b), wherein the identified difference indicates that the subject has said prostate cancer, or indicates that the subject has a risk of developing said prostate cancer, or is indicative of the prognosis of said prostate cancer in the subject, respectively.
The present invention also relates to a method for assessing the efficacy of a treatment for prostate cancer in a subject, or for monitoring the progression or regression of a prostate cancer in a subject, or for identifying a compound capable of ameliorating or treating prostate cancer in a subject, comprising: (a) measuring the levels of ten or more markers selected from the group of markers identified in Table 1 as Signature 1 markers in a population of the subject's macrophage cells or neutrophil cells before the treatment, or at a first time point, or before administering the compound to the subject, respectively; (b) measuring the levels of the ten or more selected Signature 1 markers in a population of the subject's non-phagocytic cells before the treatment, or at the first time point, or before administering the compound to the subject, respectively; (c) identifying a first difference between the measured levels of the ten or more selected Signature 1 markers in steps (a) and (b); (d) measuring the levels of the ten or more selected Signature 1 markers in a population of the subject's macrophage cells or neutrophil cells after the treatment, or at a second time point, or after the administration of the compound, respectively; (e) measuring the levels of the ten or more selected Signature 1 markers in a population of the subject's non-phagocytic cells after the treatment, or at a second time point, or after the administration of the compound, respectively; (f) identifying a second difference between the measured levels of the ten or more selected Signature 1 markers in steps (d) and (e); and (g) identifying a difference between the first difference and the second difference, wherein the difference identified in (g) is indicative of the efficacy of the treatment for said prostate cancer in the subject, or is indicative of the progression or regression of said prostate cancer in the subject, or indicates that the compound is capable of ameliorating or treating said prostate cancer in the subject, respectively.
According to a preferred embodiment the method further comprises measuring at least one standard parameter associated with said prostate cancer. The standard parameter is preferably selected from the group consisting of tumor stage, tumor grade, tumor size, tumor visual characteristics, tumor growth, tumor thickness, tumor progression, tumor metastasis tumor distribution within the body, odor, molecular pathology, genomics, or tumor angiograms.
According to a further preferred embodiment the measured levels are gene expression levels.

Brief Description of the D rawings

Figure 1 depicts microarray results in a search for a signature of normal (N) vs. aggressive (A) cancer in subtraction-normalized expression data collected from blood samples processed 4 hours after collection.
Figure 2 depicts microarray results in search for a signature of normal (N) vs. aggressive (A) cancer in subtraction-normalized expression data collected from blood samples processed 4 hours after collection. The assay was used to search for stable transcripts (defined as those in the top 1,000 differentially expressed genes, with subtraction normalization at 4 hours) that had an expression level ratio of 0.8 to 1.25 in samples processed 48 hours after collection compared to 4 hours after collection.

Detailed Description

The present disclosure provides biological markers and methods of using them to detect a cancer. More specifically, the present disclosure provides biomarkers that are specific for prostate cancer.
As used here in, a "biomarker" or "marker" refers to an analyte (e.g., a nucleic acid, DNA, RNA, peptide, protein, or metabolite) that can be objectively measured and evaluated as an indicator for a biological process. In some implementations, a marker is differentially detectable in phagocytes and is indicative of the presence or absence of prostate cancer. An analyte is differentially detectable if it can be distinguished quantitatively or qualitatively in phagocytes compared to a control, e.g., a normal or healthy control or non-phagocytic cells.
The present disclosure is based on the discovery that one or more markers selected from Tables 1 and/or 2 and/or 3 and/or 4 (Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers, respectively) are useful in diagnosing prostate cancer. By measuring the levels of the biomarkers (e.g., gene expression levels, protein expression levels, or protein activity levels) in a population of phagocytes (e.g., macrophage or neutrophils) from a human subject, one can provide a reliable diagnosis for prostate cancer.
As used herein, a "level" of a marker of this disclosure can be qualitative (e.g., presence or absence) or quantitative (e.g., amounts, copy numbers, or dosages). In some implementations, a level of a marker at a zero value can indicate the absence of this marker. The levels of any marker of this disclosure can be measured in various forms. For example, the level can be a gene expression level, a RNA transcript level, a protein expression level, a protein activity level, an enzymatic activity level.
The markers of this disclosure can be used in methods for diagnosing or aiding in the diagnosis of prostate cancer by comparing levels (e.g., gene expression levels, or protein expression levels, or protein activities) of one or more prostate cancer markers (e.g., nucleic acids or proteins) between phagocytes (e.g., macrophages or neutrophils) and non-phagocytic cells taken from the same individual. This disclosure also provides methods for assessing the risk of developing prostate cancer, prognosing said cancer, monitoring said cancer progression or regression, assessing the efficacy of a treatment, or identifying a compound capable of ameliorating or treating said cancer.
The methods of this disclosure can be applied to prostate cancer. As used herein, "prostate cancer" means any cancer of the prostate including, but not limited to, adenocarcinoma and small cell carcinoma.
In a first aspect, the methods (e.g., diagnosis of prostate cancer, prognosis of prostate cancer, or assessing the risk of developing prostate cancer) provided in the disclosure comprise: a) measuring the levels of one or more markers selected from Tables 1 and/or 2 and/or 3 and/or 4 (Signature 1 markers and/or Signature 2 markers and/or Signature 3 markers and/or Signature 4 markers, respectively) in a population of a subject's macrophage cells; b) measuring the levels of one or more of the selected markers in a population of a subject's non- phagocytic cells (e.g., T-cells, B-cells, null cells, basophils or the mixtures of two more non-phagocytic cells); comparing the measured levels in step a) to the measured levels in step b) and further identify a difference between the measured levels of a) and b). The identified difference is indicative of the diagnosis (e.g., presence or absence), prognosis (e.g., lethal outcome, or tumor stage), or the risk of developing prostate cancer.
In the first aspect, the selected markers comprise one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, fifty, one-hundred, one-hundred seventeen, or any increment between those values) markers of Signature 1, or one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen) markers of Signature 2, or one or more (e.g., two, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen) markers of Signature 3, or one or more (e.g., two, two, three, four, five, six, seven, eight, nine, or ten) markers of Signature 4. In some implementations, the selected markers are up-regulated (see Tables 1 and/or 2 and/or 3 and/or 4 for up- regulated markers) in prostate cancer patients. In some implementations, the selected markers are down-regulated (see Table 1 for down-regulated markers) in prostate cancer patients. In some implementations, the selected markers comprise at least one Signature 1 marker that is up-regulated and at least one Signature 1 marker that is down-regulated.
In a second aspect, the methods provided in this disclosure for assessing the efficacy of a treatment for prostate cancer, monitoring the progression or regression of prostate cancer, or identifying a compound capable of ameliorating or treating prostate cancer, respectively, in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 (Tables 1 and/or 2 and/or 3 and/or 4, respectively) in a population of the subject's macrophage cells before the treatment, or at a first time point, or before administration of the compound, respectively; b) measuring the levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in a population of the subject's non-phagocytic cells before the treatment, or at the first time point, or before administration of the compound, respectively; c) identifying a first difference between the measured levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in steps a) and b); d) measuring the levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in a population of the subject's macrophage cells after the treatment, or at a second time point, or after administration of the compound, respectively; e) measuring the levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in a population of the subject's non-phagocytic cells after the treatment, or at the second time point, or after administration of the compound, respectively; f) identifying a second difference between the measured levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in steps d) and e); and g) identifying a difference between the first difference and the second difference, wherein the difference identified in g) is indicative of the efficacy of the treatment for the prostate cancer, or the progression or regression of the prostate cancer, or whether the compound is capable of ameliorating or treating the prostate cancer, respectively, in the subject.
In a third aspect, the methods provided in this disclosure for assessing the efficacy of a treatment for prostate cancer, monitoring the progression or regression of prostate cancer, or identifying a compound capable of ameliorating or 15treating prostate cancer, respectively, in a subject comprising: a) measuring the levels of one or more markers selected from the group consisting of Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 (Tables 1 and/or 2 and/or 3 and/or 4, respectively) in a population of the subject's macrophage cells before the treatment, or at a first time point, or before administration of the compound, respectively; b) identifying a first difference between the measured levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in step (a) and the levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in a control (e.g., a healthy control cell, or a control cell from a healthy subject) before the treatment, or at the first time point, or before administration of the compound, respectively; c) measuring the levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in a population of the subject's macrophage cells after the treatment, or at a second time point, or after administration of the compound, respectively; d) identifying a second difference between the measured levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in step c) and the levels of the one or more selected Signature 1 and/or Signature 2 and/or Signature 3 and/or Signature 4 markers in a control after the treatment, or at the second time point, or after administration of the compound, respectively; and e) identifying a difference between the first difference and the second difference, wherein the difference identified in e) is indicative of the efficacy of the treatment for the prostate cancer, or the progression or regression of the prostate cancer, or whether the compound is capable of ameliorating or treating the prostate cancer, respectively, in the subject.
In the second and third aspects, the selected markers comprise one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, fifty, one-hundred, one-hundred seventeen, or any increment between those values) of Signature 1, or one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen) of Signature 2, or one or more (e.g., two, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen) markers of Signature 3, or one or more (e.g., two, two, three, four, five, six, seven, eight, nine, or ten) markers of Signature 4. In some implementations, the selected markers are upregulated (see Tables 1 and/or 2 and/or 3 and/or 4 for up-regulated markers) in prostate cancer patients. In some implementations, the selected markers are down-regulated (see Table 1 for down-regulated markers) in prostate cancer patients. In some implementations, the selected markers comprise at least one Signature 1 marker that is up-regulated and at least one Signature 1 marker that is down-regulated.
In some implementations, two sub-populations of phagocytic cells arc used in the methods of this disclosure, i.e., phagocytic cells that have a DNA content greater than 2n (the >2n phagocytic cells) and phagocytic cells that have a DNA content of2n (the =2n phagocytic cells). In those implementations, the levels of the selected markers in the >2n phagocytic cells are compared to the =2n phagocytic cells to identify one or more difference. The identified differences indicate whether the subject has prostate cancer, or has a risk of developing prostate cancer, or has a progressing or progressive prostate cancer.
In some implementations, the levels of one or more markers from Tables 1 or 2 or 3 or 4 are measured in combination with measuring the levels of one or more markers from a different table from among Tables 1, 2, 3, and 4.
In various implementations of the present disclosure, at least one or more of the selected markers (Signature 1 markers or Signature 2 markers or Signature 4 markers or Signature 4 markers) may be used in addition to a biological marker different from any of the selected markers. In some implementations, such biological markers may be known markers for prostate cancer. In some implementations, such biological markers and the substituted selected markers may belong to the same signaling or biological pathway (e.g., a protein synthesis pathway, Thl cytokine production pathway, transcription pathway, programmed cell death pathway), or may have similar biological function or activity (e.g., protein synthesis, Thl cytokine production, nucleotide binding, protein binding, transcription, a receptor for purines coupled to G-proteins, inhibition of programmed cell death, neutrophil activation, an IL-8 receptor, an HSP70-interacting protein, stimulating ATPase activity), or may be regulated by a common protein, or may belong to the same protein complex (e.g., an HSP70 protein complex).
In various implementations of the present disclosure, a population of the subject's macrophage cells is used as the selected phagocytic cells for measuring the levels of the selected markers (e.g., Signature 1 and/or Signature 2 markers and/or Signature 3 markers and/or Signature 4 markers) and a population of the subject's T-cells is used as the selected non-phagocytic cells for measuring the levels of the selected markers (e.g., Signature 1 and/or Signature 2 markers and/or Signature 3 markers and/or Signature 4 markers).
The gene names/descriptions provided in Tables 1 and 2 and 3 and 4 are merely illustrative. The markers of this disclosure encompass all forms and variants of any specifically described markers, including, but not limited to, polymorphic or allelic variants, isoforms, mutants, derivatives, precursors including nucleic acids and pro-proteins, cleavage products, and structures comprised of any of the markers as constituent subunits of the fully assembled structure.
A "patient", "subject", or "individual" arc used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).
As used herein, the terms "normal control", "healthy control", and "not- diseased cells" likewise mean a sample (e.g., cells, serum, tissue) taken from a source (e.g., subject, control subject, cell line) that does not have the condition or disease being assayed and therefore may be used to determine the baseline for the condition or disorder being measured. A control subject refers to any individual that has not been diagnosed as having the disease or condition being assayed. It is also understood that the control subject, normal control, and healthy control, include data obtained and used as a standard, i.e. it can be used over and over again for multiple different subjects. In other words, for example, when comparing a subject sample to a control sample, the data from the control sample could have been obtained in a different set of experiments, for example, it could be an average obtained from a number of healthy subjects and not actually obtained at the time the data for the subject was obtained.
The term "diagnosis" as used herein refers to methods by which the skilled artisan can estimate and/or determine whether or not a patient is suffering from a given disease or condition. In some implementations, the term "diagnosis" also refers to staging (e.g., Stage I, II, III, or IV) of cancer. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, e.g., a marker, the presence, absence, amount, or change in amount of which is indicative of the presence, severity, or absence of the condition.
The term "prognosis" as used herein refers to is used herein to refer to the likelihood of prostate cancer progression, including recurrence of prostate cancer, and/or evaluation of whether the prostate cancer is aggressive or indolent.
Each implementation described herein may be combined with any other implementation described herein.
Methods using the prostate cancer markers described herein provide high specificity, sensitivity, and accuracy in detecting and diagnosing prostate cancer. The methods also eliminate the "inequality of baseline" that is known to occur among individuals due to intrinsic (e.g., age, gender, ethnic background, health status and the like) and temporal variations in marker expression. Additionally, by using a comparison of phagocytes and non-phagocytes from the same individual, the methods also allow detection, diagnosis, and treatment to be personalized to the individual. Accordingly, in some implementations, the disclosure provides non-invasive assays for the early detection of prostate cancer, i.e., before the prostate cancer can be diagnosed by convention a diagnostic techniques, e.g., imaging techniques, and, therefore, provide a foundation for improved decision-making relative to the needs and strategies for intervention, prevention, and treatment of individuals with such disease or condition.
The methods described herein are supported by whole genome microarray data of total RNA samples isolated from macrophages and from non-phagocytic T cells. The samples were obtained from human subjects with and without prostate cancer. The data from these microarray experiments demonstrate that macrophagc-T cell comparisons easily and accurately differentiate between prostate cancer patients and human subjects without prostate cancer.
The methods of this disclosure can be used together with any known diagnostic methods, such as physical inspection, visual inspection, biopsy, scanning, histology, radiology, imaging, ultrasound, use of a commercial kit, genetic testing, immunological testing, analysis of bodily fluids, or monitoring neural activity.
Phagocytic cells that can be used in the methods of this disclosure include all types of cells that are capable of ingesting various types of substances (e.g., apoptotic cells, infectious agents, dead cells, viable cells, cell-free DNAs, cell-free RNAs, cell-free proteins). In some implementations, the phagocytic cells are neutrophils, macrophages, monocytes, dendritic cells, foam cells, mast cells, eosinophils, or keratinocytes. In some implementations, the phagocytic cells can be a mixture of different types of phagocytic cells. In some implementations, the phagocytic cells can be activated phagocytic cells, e.g., activated macrophages or neutrophils. In some implementations, a phagocyte is a histiocyte, e.g., a Langerhans cell.
As used herein, "treating" prostate cancer refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms associated with diseases or conditions.
As used herein, "administering" or "administration of a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitonealy, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorbtion, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow, or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some aspects, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient. In some implementations, a compound or an agent is administered orally, e.g., to a subject by ingestion, or intravenously, e.g., to a subject by injection. In some implementations, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
In certain implementations, markers used in the methods of disclosure are up-regulated or activated in phagocytes (e.g., macrophages or neutrophils) compared to non-phagocytes. In certain implementations, markers used in the methods of disclosure are down-regulated or inhibited in phagocytes (e.g., macrophages or neutrophils) compared to non-phagocytes. As used herein, "up-regulation or up-regulated" can refer to an increase in expression levels (e.g., gene expression or protein expression), gene copy numbers, gene dosages, and other qualitative or quantitative detectable state of the markers. Similarly, "down- regulation or down-regulated" can refer to a decrease in expression levels, gene copy numbers, gene dosages, and other qualitative or quantitative detectable state of the markers. As used herein, "activation or activated" can refer to an active state of the marker, e.g., a phosphorylation state, a DNA methylation state, or a DNA acetylation state. Similarly, "inhibition or inhibited" can refer to a repressed state or an inactivated state of the marker, e.g., a de-phosphorylation state, a ubiquitination state, or a DNA de-methylation state.
In certain implementations, methods of this disclosure also comprise at least one of the following steps before determination of various levels: i) lysing the phagocytic or non-phagocytic cells; and ii) extracting cellular contents from the lysed cells. Any known cell lysis and extraction methods can be used herein. In certain implementations, at least one or more prostate cancer markers are present in the phagocytes. In certain implementations, there is no marker present in the cellular contents of the non-phagocytic cells.
In certain implementations, the phagocytic cells and/or non-phagocytic cells are isolated from a bodily fluid sample, tissues, or population of cells. Exemplary bodily fluid samples can be whole blood, urine, stool, saliva, lymph fluid, cerebrospinal fluid, synovial fluid, cystic fluid, ascites, pleural effusion, fluid obtained from a pregnant woman in the first trimester, fluid obtained from a pregnant woman in the second trimester, fluid obtained from a pregnant woman in the third trimester, maternal blood, amniotic fluid, chorionic villus sample, fluid from a preimplantation embryo, maternal urine, maternal saliva, placental sample, fetal blood, lavage and cervical vaginal fluid, interstitial fluid, buccal swab sample, sputum, bronchial lavage, Pap smear sample, or ocular fluid. In some implementations, the phagocytic cells or non-phagocytic cells are isolated from white blood cells.
In the methods of this disclosure, cell separation/isolation/ purification methods are used to isolate populations of cells from bodily fluid sample, cells, or tissues of a subject. A skilled worker can use any known cell separation/isolation/purification techniques to isolate phagocytic cells and non-phagocytic cells from a bodily fluid. Exemplary techniques include, but are not limited to, using antibodies, flow cytometry, fluorescence activated cell sorting, filtration, gradient-based centrifugation, elution, microfluidics, magnetic separation technique, fluorescent-magnetic separation technique, nanostructure, quantum dots, high throughput microscope-based platform, or a combination thereof.
In certain implementations, the phagocytic cells and/or non-phagocytic Cells are isolated by using a product secreted by the cells. In certain implementations, the phagocytic cells and/or non-phagocytic cells are isolated by using a cell surface target (e.g., receptor protein) on the surface of the cells. In some implementations, the cell surface target is a protein that has been engulfed by phagocytic cells. In some implementations, the cell surface target is expressed by cells on their plasma membranes. In some implementations, the cell surface target is an exogenous protein that is translocated on the plasma membranes, but not expressed by the cells (e.g., the phagocytic cells). In some implementations, the cell surface target is a marker of prostate cancer.
In certain aspects of the methods described herein, analytes include nucleic acids, proteins, or any combinations thereof. In certain aspects of the methods described herein, markers include nucleic acids, proteins, or any combinations thereof. As used herein, the term "nucleic acid" is intended to include DNA molecules (e.g., eDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA-RNA hybrids, and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be a nucleotide, oligonucleotide, double-stranded DNA, single-stranded DNA, multi-stranded DNA, complementary DNA, genomic DNA, non-coding DNA, messenger RNA (mRNAs), microRNA (miRNAs), small nucleolar RNA (snoRNAs), ribosomal RNA (rRNA), transfer RNA (tRNA), small interfering RNA (siRNA), heterogeneous nuclear RNAs (hnRNA), or small hairpin RNA (shRNA). In some implementations, the nucleic acid is a transrenal nucleic acid. A transrenal nucleic acid is an extracellular nucleic acid that is excreted in the urine. See, e.g., U.S. Patent Publication No. 20100068711 and U.S. Patent Publication No. 20120021404 .
As used herein, the term "amino acid" includes organic compounds containing both a basic amino group and an acidic carboxyl group. Included within this term are natural amino acids (e.g., L-amino acids), modified and unusual amino acids (e.g., D-amino acids and -amino acids), as well as amino acids which are known to occur biologically in free or combined form but usually do not occur in proteins. Natural protein occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, tryptophan, proline, and valine. Natural non-protein amino acids include arginosuccinic acid, citrulline, cysteine sulfuric acid, 3,4-dihydroxyphenylalanine, homocysteine, homoserine, ornithine, 3-monoiodotyrosine, 3,5-diiodotryosine, 3, 5, 5-triiodothyronine, and 3,3',5,5'- tetraiodothyronine. Modified or unusual amino acids include D-amino acids, hydroxylysine, 4-hydroxyproline, N-Cbz- protected amino acids, 2,4-diaminobutyric acid, homoarginine, norleucine, N-methylaminobutyric acid, naphthylalanine, phenylglycine, a-phenylproline, tert- leucine, 4-aminocyclohexylalanine, N-methylnorleucine, 3 ,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidine-4- carboxylic acid, 6-aminocaproic acid, trans-4-(aminomethyl)- cyclohexanecarboxylic acid, 2-, 3-, and 4-(aminomethyl)- benzoic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, and 2- benzyl-5-aminopentanoic acid.
As used herein, the term "peptide" includes compounds that consist of two or more amino acids that arc linked by means of a peptide bond. Pcptidcs may have a molecular weight of less than 10,000 Daltons, less than 5,000 Daltons, or less than 2,500 Daltons. The term "peptide" also includes compounds containing both peptide and non-peptide components, such as pseudopeptide or peptidomimetic residues or other non-amino acid components. Such compounds containing both peptide and non-peptide components may also be referred to as a "peptide analog."
As used herein, the term "protein" includes compounds that consist of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Proteins used in methods of the disclosure include, but are not limited to, amino acids, peptides, antibodies, antibody fragments, cytokines, lipoproteins, or glycoproteins.
As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full length antibodies which have an 5 immunoglobulin Fe region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules, and antibody fragments (e.g., Fab or F(ab')2, and Fv). For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.
As used herein, the term "cytokine" refers to a secreted protein or active fragment or mutant thereof that modulates the activity of cells of the immune system. Examples of cytokines include, without limitation, interleukins, interferons, chemokines, tumor necrosis factors, colonystimulating factors for immune cell precursors, and the like.
As used herein, the term "lipoprotein" includes negatively charged compositions that comprise a core of hydrophobic cholesteryl esters and triglyceride surrounded by a surface layer of amphipathic phospholipids with which free cholesterol and apolipoproteins are associated. Lipoproteins may be characterized by their density (e.g. very-low-density lipoprotein (VLDL), low- density lipoprotein (LDL) and high density lipoprotein (HDL)), which is determined by their size, the relative amounts of lipid and protein. Lipoproteins may also be characterized by the presence or absence of particular modifications (e.g. oxidization, acetylation, or glycation).
As used herein, the term "glycoprotein" includes glycosides which have one or more oligo- or polysaccharides covalently attached to a peptide or protein. Exemplary glycoproteins can include, without limitation, immunoglobulins, members of the major histocompatibility complex, collagens, mucins, glycoprotein IIb/IIIa, glycoprotein-41 (gp41) and glycoprotein-120 gp12), follicle-stimulating hormone, alpha-fetoprotein, erythropoietin, transferrins, alkaline phosphatase, and lectins.
In some implementations of the disclosure, a sample may comprise one or more stabilizers for a cell or an analyte such as DNA, RNA, and/or protein. For example, a sample may comprise a DNA stabilizer, an RNA stabilizer, and/or a protein stabilizer. Stabilizers are well known in the art and include, for example, DNAse inhibitors, RNAse inhibitors, and protease inhibitors or equivalents thereof.
In some implementations of the disclosure, levels of at least one or more prostate cancer markers are compared. This comparison can be quantitative or qualitative. Quantitative measurements can be taken using any of the assays described herein. For example, sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, targeted sequencing, whole- genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single- base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD^® sequencing, MS-PET sequencing, mass spectrometry, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, clectrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorptionlionization-time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), polymerase chain reaction (PCR) analysis, quantitative PCR, real-time PCR, fluorescence assay, colorimetric assay, chemiluminescent assay, or a combination thereof.
Quantitative comparisons can include statistical analyses such as t-tcst, ANOVA, Krustai-Wallis, Wilcoxon, Mann-Whitney, and odds ratio. Quantitative differences can include differences in the levels of markers between levels or differences in the numbers of markers present between levels, and combinations thereof. Examples of levels of the markers can be, without limitation, gene expression levels, nucleic acid levels, and protein levels. Qualitative differences can include, but are not limited to, activation and inactivation, protein degradation, nucleic acid degradation, and covalent modifications.
In certain implementations of the disclosure, the level is a nucleic acid Level or a protein level, or a combination thereof. The level can be qualitatively or quantitatively determined.
A nucleic acid level can be, without limitation, a genotypic level, a single nucleotide polymorphism level, a gene mutation level, a gene copy number level, a DNA methylation level, a DNA acetylation level, a chromosome dosage level, a gene expression level, or a combination thereof.
The nucleic acid level can be determined by any methods known in the art to detect genotypes, single nucleotide polymorphisms, gene mutations, gene copy numbers, DNA methylation states, DNA acetylation states, chromosome dosages. Exemplary methods include, but are not limited to, polymerase chain reaction (PCR) analysis, sequencing analysis, electrophoretic analysis, restriction fragment length polymorphism (RFLP) analysis, Northern blot analysis, quantitative PCR, reverse-transcriptase-PCR analysis (RT-PCR), allele-specific oligonucleotide hybridization analysis, comparative genomic hybridization, heteroduplex mobility assay (HMA), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophisis (DGGE), RNAase mismatch analysis, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ ionization-time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), surface plasmon resonance, Southern blot analysis, in situ hybridization, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), microarray, comparative genomic hybridization, karyotyping, multiplex ligation-dependent probe amplification (MLPA), Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF), microscopy, methylation specific PCR (MSP) assay, Hpall tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay, radioactive acetate labeling assays, colorimetric DNA acetylation assay, chromatin immunoprecipitation combined with microarray (ChiP-on-chip) assay, restriction landmark genomic scanning, Methylated DNA immunoprecipitation (MeDIP), molecular break light assay for DNA adenine methyltransferase activity, chromatographic separation, methylation-sensitive restriction enzyme analysis, bisulfite-driven conversion of non-methylated cytosine to uracil, co-amplification at lower denaturation temperature-PeR (COLD-PCR), multiplex PCR, methyl-binding PCR analysis, or a combination thereof.
As used herein, the term "sequencing" is used in a broad sense and refers to any technique known in the art that allows the order of at least some consecutive nucleotides in at least part of a nucleic acid to be identified, including without limitation at least part of an extension product or a vector insert. Exemplary sequencing techniques include targeted sequencing, single molecule real-time sequencing, whole transcriptome shotgun sequencing ("RNA-seq"), electron microscopy-based sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, exon sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high- throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD^® sequencing, MS-PET sequencing, mass spectrometry, and a combination thereof. In some implementations, sequencing comprises an detecting the sequencing product using an instrument, for example but not limited to an ABI PRISM^® 377 DNA Sequencer, an ABI PRISM^® 310, 3100, 3100-Avant, 3730, or 3730x1 Genetic Analyzer, an ABI PRISM^® 3700 DNA Analyzer, or an Applied Biosystems SOLiDTM System (all from Applied Biosystems), a Genome Sequencer 20 System (Roche Applied Science), or a mass spectrometer. In certain implementations, sequencing comprises emulsion PCR. In certain implementations, sequencing comprises a high throughput sequencing technique, for example but not limited to, massively parallel signature sequencing (MPSS).
In further implementations of the disclosure, a protein level can be a protein expression level, a protein activation level, or a combination thereof. In some implementations, a protein activation level can comprise determining a phosphorylation state, an ubiquitination state, a myristoylation state, or a conformational state of the protein.
A protein level can be detected by any methods known in the art for detecting protein expression levels, protein phosphorylation state, protein ubiquitination state, protein myristoylation state, or protein conformational state. In some implementationsimplementations, a protein level can be determined by an immunohistochemistry assay, an enzymelinked immunosorbent assay (ELISA), in situ hybridization, chromatography, liquid chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC), gas chromatography, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser deorption/ionization-time of flight (SELDI-TOF) mass spectrometry, quadrupole- time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), radioimmunoassays, microscopy, microfluidic chip-based assays, surface plasmon resonance, sequencing, Western blotting assay, or a combination thereof.
As used herein, the "difference" between different levels detected by the methods of this disclosure can refer to different gene copy numbers, different DNA, RNA, or protein expression levels, different DNA methylation states, different DNA acetylation states, and different protein modification states. The difference can be a difference greater than 1 fold. In some implementations, the difference is a 1.05-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold difference. In some implementations, the difference is any fold difference between 1-10, 2-10, 5-10, 10-20, or 10-100 fold.
In some implementationsimplementations, the difference is differential gene expression (DGE), e.g. DGE of phagocytes vs. non-phagocytes. DGE can be measured as X = logz(Yp) logz(YNP). The DGE may be any number, provided that it is significantly different between the phagocytes and the non-phagocytes. For example, a 2-fold increased in gene expression could be represented as X log₂(Yr) -log₂(YNr) = log₂(YrNKr) =log₂(2) = 1, while a 2-fold decrease in gene expression could be represented as X = logz(Yp) - logz(YNP) = logz(Yr/Y NP)=logz(1/2) = -1. Down-regulated genes have X < 0, while up-regulated genes have X> 0. See, e.g., Efron, J Am Stat Assoc 104:1015-1028 (2009).
A general principle of assays to detect markers involves preparing a sample or reaction mixture that may contain the marker (e.g., one or more of DNA, RNA, or protein) and a probe under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.
For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction. In one implementation of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support. In another implementation, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.
There are many established methods for anchoring assay components to A solid phase. These include, without limitation, marker or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain implementations, the surfaces with immobilized assay components can be prepared in advance and stored.
Other suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the marker or probe belongs. Well known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
In order to conduct assays with the above mentioned approaches, the non- immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.
In certain exemplary implementations, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.
It is also possible to directly detect marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (see, for example, U.S. Patent Nos. 5,631,169 and 4,868,103 ). A fluorophore label on the first, 'donor' molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. A FRET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
In another implementations, determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C, 1991, Anal. Chern. 63:2338 2345 and Szabo et al, 1995, Curr. Opin. Struct. Biol. 5:699 705). As used herein, "BIA" or "surface plasmon resonance" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.
Alternatively, in another implementations, analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase. In such an assay, the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, marker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (sec, for example, Rivas and Minton (1993) Trends Biochem. Sci. 18:284). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the marker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example 10 through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard (1998) J. Mol. Recognit. 11 :141; Hage and Tweed (1997) J. Chromatogr. B. Biomed. Sci. Appl. 12:499). Gel electrophoresis may also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typically preferred. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.
In certain exemplary implementations, the level of mRNA corresponding to the marker can be determined either by in situ and/or by in vitro formats in a biological sample using methods known in the art. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from blood cells (see, e.g., Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987 1999). Additionally, large numbers of cells and/or samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski ( 1989, U.S. Patent No. 4,843,155 ).
Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. In certain exemplary implementations, a diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length eDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a marker of the present disclosure. Other suitable probes for use in the diagnostic assays of the disclosure are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.
In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in a gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present disclosure.
An alternative method for determining the level of mRNA corresponding to a marker of the present disclosure in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental implementation set forth in U.S. Patent Nos. 4,683,195 and 4,683,202 ), COLD-PCR (Li etal. (2008) Nat. Med. 14:579), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA,88:189), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication ( U.S. Patent No. 5,854,033 ) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers arc defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
For in situ methods, mRNA does not need to be isolated from the sample (e.g., a bodily fluid (e.g., blood cells)) prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker.
As an alternative to making determinations based on the absolute expression level of the marker, determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell- specific genes. This normalization allows the comparison of the expression level in a patient sample from one source to a patient sample from another source, e.g., to compare a population of phagocytic from an individual to a population of non-phagocytic cells from the individual.
In one implementation of this disclosure, a protein or polypeptide corresponding to a marker is detected. In certain implementations, an agent for detecting a protein or polypeptide can be an antibody capable of binding to the polypeptide, such as an antibody with a detectable label. As used herein, the term "labeled," with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. In one format, antibodies, or antibody fragments, can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, magnetite and the like.
A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, competitive and non-competitive immunoassay, enzyme immunoassay (EIA), radioimmunoassay (RIA), antigen capture assays, two-antibody sandwich assays, Western blot analysis, enzyme linked immunoabsorbant assay (ELISA), a planar array, a colorimetric assay, a chemiluminescent assay, a fluorescent assay, and the like. Immunoassays, including radioimmmunoassays and enzyme- linked immunoassays, are useful in the methods of the present disclosure. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells (e.g., bodily fluid cells such as blood cells) express a marker of the present disclosure.
One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present disclosure. For example, protein isolated from cells (e.g., bodily fluid cells such as blood cells) can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.
In certain exemplary implementations, assays arc provided for diagnosis, prognosis, assessing the risk of developing prostate cancer, assessing the efficacy of a treatment, monitoring the progression or regression of prostate cancer, and identifying a compound capable of ameliorating or treating prostate cancer. An exemplary method for these methods involves obtaining a bodily fluid sample from a test subject, isolating phagocytes and non-phagocytes, and contacting the phagocytes and non-phagocytes with a compound or an agent capable of detecting one or more of the markers of the disease or condition, e.g., marker nucleic acid (e.g., mRNA, genomic DNA), marker peptide (e.g., polypeptide or protein), marker lipid (e.g., cholesterol), or marker metabolite (e.g., creatinine) such that the presence of the marker is detected. In one implementation, an agent for detecting marker mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to marker mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length marker nucleic acid or a portion thereof. Other suitable probes for use in the diagnostic assays of the disclosure are described herein.
As used herein, a compound capable of ameliorating or treating prostate cancer can include, without limitations, any substance that can improve symptoms or prognosis, prevent progression of the prostate cancer, promote regression of the prostate cancer, or eliminate the prostate cancer.
The methods of the disclosure can also be used to detect genetic alterations in a marker gene, thereby determining if a subject with the altered gene is at risk for developing prostate cancer characterized by misregulation in a marker protein activity or nucleic acid expression. In certain implementations, the methods include detecting, in phagocytes, the presence or absence of a genetic alteration characterized by an alteration affecting the integrity of a gene encoding a marker peptide and/or a marker gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from one or more marker genes; 2) an addition of one or more nucleotides to one or more marker genes; 3) a substitution of one or more nucleotides of one or more marker genes, 4) a chromosomal rearrangement of one or more marker genes; 5) an alteration in the level of a messenger RNA transcript of one or more marker genes; 6) aberrant modification of one or more marker genes, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of one or more marker genes; 8) a non-wild type level of a one or more marker proteins; 9) allelic loss of one or more marker genes; and 10) inappropriate post-translational modification of one or more marker proteins. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in one or more marker genes.
In certain implementations, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 , 4,683,202 and 5,854,033 ), such as real-time PCR, COLD-PCR (Li et al. (2008) Nat. Med. 14:579), anchor PCR, recursive PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077; Prodromou and Pearl (1992) Protein Eng. 5:827; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360), the latter of which can be particularly useful for detecting point mutations in a marker gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675). This method can include the steps of collecting a sample of cell free bodily fluid from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a marker gene under conditions such that hybridization and amplification of the marker gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874), transcriptional amplification system (Kwoh et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173), Q Beta Replicase (Lizardi et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes arc especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative implementation, mutations in one or more marker genes from a sample can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, optionally amplified, digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531 ) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other implementations, genetic mutations in one or more of the markers described herein can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al. (1996) Human Mutation 7: 244; Kozal et al. (1996) Nature Medicine 2:753). For example, genetic mutations in a marker nucleic acid can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another implementation, any of a variety of sequencing reactions known in the art can be used to directly sequence a marker gene and detect mutations by comparing the sequence of the sample marker gene with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101 ; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147).
Other methods for detecting mutations in a marker gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild- type marker sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1nuclease to enzymatically digesting the mismatched regions. In other implementations, either DNAJDNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286. In one implementation, the control DNA or RNA can be labeled for detection.
In still another implementation, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in marker cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657). According to an exemplary implementation, a probe based on a marker sequence, e.g., a wild-type marker sequence, is hybridized to a eDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039 .
In other implementation, alterations in electrophoretic mobility will be Used to identify mutations in marker genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766, see also Cotton (1993) Mutat. Res. 285:125; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73). Single-stranded DNA fragments of sample and control marker nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one implementation, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
In yet another implementation the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further implementation, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chern. 265:12753).
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki ct al. (1986) Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant disclosure. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17:2437) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain implementations amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA gg:1g9). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
An exemplary method for detecting the presence or absence of an analyte (e.g., DNA, RNA, protein, polypeptide, or the like) corresponding to a marker of the disclosure in a biological sample involves obtaining a bodily fluid sample (e.g., blood) from a test subject and contacting the bodily fluid sample with a compound or an agent capable of detecting one or more markers. Detection methods described herein can be used to detect one or more markers in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a polypeptide corresponding to a marker of the disclosure include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a polypeptide corresponding to a marker of the disclosure include introducing into a subject a labeled antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Because each marker is also an analyte, any method described herein to detect the presence or absence of a marker can also be used to detect the presence or absence of an analyte.
The markers useful in the methods of the disclosure can include any mutation in any one of the markers. Mutation sites and sequences can be identified, for example, by databases or repositories of such information, e.g., The Human Gene Mutation Database (www.hgmd.cf.ac.uk), the Single Nucleotide Polymorphism Database (dbSNP, www.ncbi.nlm.nih.gov/projects/SNP), and the Online Mendelian Inheritance in Man (OMTM) website (www.ncbi.nlm.nih.gov/omim).
The present disclosure also provides kits that comprise marker detection agents that detect at least one or more of the prostate cancer markers described herein.
The present disclosure also provides methods of treating or preventing prostate cancer in a subject comprising administering to said subject an agent that modulates the activity or expression or disrupts the function of at least one or more of the markers of this disclosure.
The one or more markers identified by this disclosure (e.g., markers in Tables 1 and 2 and 3 and 4) may be used in the treatment of prostate cancer. For example, a marker (e.g., a protein or gene) identified by the present disclosure may be used as a molecular target for a therapeutic agent. A marker identified by the disclosure also may be used in any of the other methods of the disclosure, e.g., for monitoring the progression or regression of a disease or condition. In certain implementations, the one or more markers identified by the methods of this disclosure may have therapeutic potential. For example, if a marker is identified as being up-regulated (or down-regulated), see, for example, the up-regulated (or down- regulated) markers in Tables 1 and 2 and 3 and 4, or activated (or inhibited) in phagocytic cells from a subject having prostate cancer, a compound or an agent that is capable of down-regulating (or up-regulating) or inhibiting (or activating) said marker may be useful in treating prostate cancer. Similarly, a gene protein expression level, a protein expression level, or a combination thereof may be useful in this aspect of the disclosure.
Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.
Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).
The singular forms "a," "an," and "the" include the plurals unless the context clearly dictates otherwise.
The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.
It is to be understood that the implementations of the present disclosure which have been described are merely illustrative of some of the applications of the principles of the present disclosure.
The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the disclosure as these and other equivalent implementations will be apparent in view of the present disclosure and accompanying claims.

Examples

Example 1: Identification of Signature 1

A microarray was used to search for a signature of normal vs. aggressive cancer in subtraction-normalized expression data collected from blood samples processed 4 hours after collection (Figure 1). Based on this assay, the 117 genes of Signature 1 were selected and cross validated as described in Example 5, below. Signature 1 had a sensitivity (probability of correctly detecting aggressive cancer) of 61%, and a specificity (probability of correctly identifying normal patients) of 58%.

Table 1: Signature 1

Signature 1 marker #	Illumina Transcri pt ID	Gene name	Aggressive cancer mean	Normal mean	Upregulated (UR) or downregulated (DR) in aggressive cancer
1	15689	OAS3	-9.0142958	-8.6439767	Cancer DR
2	14409	RPL13L	-1.2329318	-1.0853327	Cancer DR
3	20119	RDH5	29.3184458	29.7644395	Cancer DR
4	18791	SOX6	-1.9161712	-1.7990754	Cancer DR
5	10951	OSBPL1A	-35.893494	-48.758773	Cancer UR
6	28512	GP5	10.9758038	3.5258523	Cancer UR
7	10695	GPR89A	10.5690901	10.9658205	Cancer DR
8	86688	HS.336511	-3.1086968	-2.8816541	Cancer DR
9	10875	TMEM80	-47.885292	-46.725592	Cancer DR
10	13640	PPPDE2	-160.41714	-159.55055	Cancer DR
11	22832	RYR3	-1.3796285	-1.265545	Cancer DR
12	168345	RHBDD2	77.0791083	79.0695935	Cancer DR
13	25536	MCIR	74.1685045	74.8626128	Cancer DR
14	15079	CCDC128	7.1783549	-54.138047	Cancer UR
15	33909	LOC285588	3.2824067	-1.8452802	Cancer UR
16	7272	ERGIC1	-76.02445	-74.709689	Cancer DR
17	177008	H2BFWT	-1.1260872	-0.9863237	Cancer DR
18	20474	CYB561	124.258067	125.519911	Cancer DR
19	41366	LOC643879	-0.1453645	0.0472667	Cancer DR
20	18811	RPL37	5.53447215	5.72682674	Cancer DR
21	180795	UGT3A2	4.67348819	-1.6783285	Cancer UR
22	2007	TP53I3	-141.98502	-140.64734	Cancer DR
23	16880	VWA5A	-19.190299	-18.947648	Cancer DR
24	123241	HS.571060	-1.460709	-1.3092629	Cancer DR
25	4653	CREB1	1888.87492	1929.09126	Cancer DR
26	18195	CYP4F2	-2.3318799	-2.2203552	Cancer DR
27	16552	TNFRSF11A	-1.1759614	-1.0106681	Cancer DR
28	5994	OAS2	-24.115038	-23.431537	Cancer DR
29	75591	HS.119922	-0.9591632	-0.8249897	Cancer DR
30	166060	FLJ39632	-0.3382191	-0.1727163	Cancer DR
31	8229	RP11-49G10.8	-6.0144268	-5.8367272	Cancer DR
32	18712	KATNAL1	4.70953157	-1.8584848	Cancer UR
33	30114	GCNT2	-0.1594717	0.0188688	Cancer DR
34	346904	LOC729212	-0.476	-0.3228795	Cancer DR
35	163101	IRF9	-392.03757	-380.96229	Cancer DR
36	33349	LOC653216	-1.0175463	-0.8626032	Cancer DR
37	42341	LOC440386	-0.8536411	-0.6714295	Cancer DR
38	6769	INF2	3.25469335	3.48877586	Cancer DR
39	15843	JARID2	-380.58538	-378.03988	Cancer DR
40	17229	HNRNPM	32.3962097	36.4189036	Cancer DR
41	1894	IL5RA	0.83011712	1.23681659	Cancer DR
42	182198	YOD1	-1.0684564	-0.968089	Cancer DR
43	21388	VHL	-153.61908	-149.34478	Cancer DR
44	11812	TRIM56	96.3762478	97.9483118	Cancer DR
45	347804	LOC10013027	29.0218497	29.6648654	Cancer DR
46	27308	UBE2I	-1.5908237	-1.459543	Cancer DR
47	11362	C80RF30A	-2.8852459	-2.7452391	Cancer DR
48	15490	METT11D1	119.293311	120.326483	Cancer DR
49	29227	ZSCAN22	0.547568	0.68218938	Cancer DR
50	16766	ZNF385B	-0.7907855	-0.6223701	Cancer DR
51	24801	C200RF94	0.40482839	0.81268385	Cancer DR
52	173747	D4S234E	531.294243	538.45889	Cancer DR
53	23467	C70RF20	-33.821791	-33.360883	Cancer DR
54	170201	CCDC88C	200.919891	203.185051	Cancer DR
55	2163	KRT38	4.69638401	-1.994684	Cancer UR
56	162122	LOC727735	3.0613806	-2.517745	Cancer UR
57	171831	LOC732215	-0.9351387	-0.8075807	Cancer DR
58	343768	LOC646353	-0.8575194	-0.7297925	Cancer DR
59	21343	DPRX	2.98927307	-2.5628675	Cancer UR
60	23908	SLC2A14	-4.387825	-4.1721651	Cancer DR
61	341890	LOC732446	-0.6644245	-0.5321704	Cancer DR
62	33455	RNF5P1	-82.512422	-80.617845	Cancer DR
63	22073	MEST	-0.6877526	-0.5880494	Cancer DR
64	338462	LOC100130829	2.19340981	-3.3179853	Cancer UR
65	31584	LOC646223	-4.695691	-4.5120316	Cancer DR
66	13066	C190RF50	-130.26601	-128.0409	Cancer DR
67	11344	VPS16	-74.322484	-71.72812	Cancer DR
68	28868	TCEALI	-2.2433124	-2.1065912	Cancer DR
69	6866	TRIM28	43.6841007	44.7414677	Cancer DR
70	138370	ANKRD13D	-54.17119	-52.791341	Cancer DR
71	180926	STX6	-190.67106	-189.13492	Cancer DR
72	28557	AGFG2	45.1171985	45.500398	Cancer DR
73	179882	IL17RD	251.863927	258.077015	Cancer DR
74	162687	BCR	8.03158781	8.21889719	Cancer DR
75	176690	SP100	-15.413932	-14.913489	Cancer DR
76	12053	RASL1OB	3.75650279	-2.8100791	Cancer DR
77	71322	HS.13291	-22.921159	-22.296491	Cancer DR
78	17081	C10RF188	16.3121173	16.6240139	Cancer DR
79	13590	NMNAT2	2.45548587	-2.5067911	Cancer DR
80	7282	CTHRC1	0.73487218	0.89298341	Cancer DR
81	18789	ASS1	-1.6801724	-1.5631152	Cancer DR
82	352616	LOC791120	163.351813	164.477126	Cancer DR
83	34136	LOC649493	-0.6295056	-0.4795787	Cancer DR
84	346345	FLJ31306	21.087961	21.6361882	Cancer DR
85	29344	VGLL4	49.4631916	50.6916039	Cancer DR
86	336512	LOC100132568	-1.6082874	-1.4598448	Cancer DR
87	6870	TCEB3C	-1.8559032	-1.7141848	Cancer DR
88	12233	SBDSP	29.0236367	29.6106915	Cancer DR
89	338504	LOC100132535	-0.0770285	0.17556409	Cancer DR
90	11118	C80RF37	609.519491	637.907911	Cancer DR
91	14405	GOLGA8B	2542.72843	2556.47364	Cancer DR
92	593	SOLH	-5.8111916	-5.2700925	Cancer DR
93	15604	LOC400464	50.0233917	50.473654	Cancer DR
94	22944	NKX6-1	2.1845754	-2.4379099	Cancer UR
95	163801	LOC442517	-0.1049745	0.0275036	Cancer DR
96	29810	ATP1B4	2.37077671	-2.1346992	Cancer UR
97	167794	USP41	-2.2619871	-2.0813413	Cancer DR
98	388123	MIR30E	-0.2759666	-0.1673701	Cancer DR
99	138008	HAND2	-0.7726292	-0.6345135	Cancer DR
100	177432	CASP7	-	-	Cancer DR
101	1696	ZNF347	3.2033801	3.4224514	Cancer DR
102	40864	LOC653598	29.521183	29.910059	Cancer DR
103	33044	LOC401238	-	-	Cancer DR
104	39554	LOC440014	-	-	Cancer DR
105	175279	LOC339352	14.598665	14.864387	Cancer DR
106	23490	NEK7	-5.929757	-	Cancer DR
107	181743	LOC646463	-	-	Cancer DR
108	15634	CCL8	-	-	Cancer DR
109	34049	LOC642196	3.68100061	-	Cancer UR
110	79403	HS.157344	10.079453	10.442659	Cancer DR
111	7196	DHX9	-	-3.934944	Cancer DR
112	44285	LOC388237	1.0386950	1.1938941	Cancer DR
113	178108	KCNJ14	0.4168120	0.5604316	Cancer DR
114	166167	OR703	-0.6721556	-	Cancer DR
115	18273	GLTSCR1	33.809635	34.504022	Cancer DR
116	1736	NOM02	-112.57512	-	Cancer DR
117	175566	WDR37	42.7679257]	46.083732	Cancer DR

Example 2: Identification of Signature 2 Z (comparative example)

A microarray was used to search for a signature of normal vs. aggressive cancer in subtraction-normalized expression data collected from blood samples processed 4 hours after collection. The assay was used to search for stable transcripts (defined as those in the top 1,000 differentially expressed genes, with subtraction normalization at 4 hours) that had an expression level ratio of 0.8 to 1.25 in samples processed 48 hours after collection compared to 4 hours after collection (Figure 2). Based on this assay, 18 genes were selected and cross validated as described in Example 5, below. Signature 2 had a sensitivity (probability of correctly detecting aggressive cancer) of 60%, and a specificity (probability of correctly identifying normal patients) of 66%.

Table 2: Signature 2

Signature 2 marker #	lllumina Transcript ID	Gene name	Aggressive cancer mean	Normal mean	Upregulated (UR) or downregulate d (DR) in aggressive
1	175412	REPS2	0.14613724	-7.5068116	Cancer DR
2	33909	LOC285588	3.2824067	-1.8452802	Cancer UR
3	22438	C200RF112	2.62604692	-4.0643154	Cancer DR
4	337363	DIPAS	1.06084697	-3.2177178	Cancer UR
5	165657	TPRX1	1.79654397	-2.8351934	Cancer UR
6	38966	FLJ16369	1.13600879	-2.5978003	Cancer UR
7	44021	LOC652255	3.03708564	-1.7358787	Cancer DR
8	41116	LOC642130	2.03361554	-1.9989049	Cancer UR
9	21343	DPRX	2.98927307	-2.5628675	Cancer UR
10	339666	LOC440225	1.72985119	-2.6607478	Cancer UR
11	91353	HS.434989	1.08668984	-2.5889165	Cancer UR
12	40621	LOC642335	2.69733599	-2.2656365	Cancer UR
13	369972	GGTLC1	1.04843521	-3.889128	Cancer DR
14	32275	LOC642966	4.72677994	-2.0890642	Cancer UR
15	22944	NKX6-1	2.1845754	-2.4379099	Cancer DR
16	17458	GPC5	0.40241623	-4.2203747	Cancer DR
17	13240	RASGEF1C	0.83201981	-3.3957263	Cancer DR
18	22752	ACOT11	2.83207957	-2.2946388	CanccrUR

Example 3: Identification of Signature 3 (comparative example)

A microarray was used as in Example 2 to search for a signature of normal vs. aggressive cancer in subtraction-normalized expression data collected from blood samples processed 4 hours after collection. Based on this assay, 18 genes were selected and cross validated as described in Example 5, below. Signature 3 had a sensitivity (probability of correctly detecting aggressive cancer) of 45%, and a specificity (probability of correctly identifying normal patients) of 64%.

Table 3: Signature 3

Signature 3 marker #	Illumina Transcript ID	Gene name	Aggressive cancer mean	Normal mean	Upregulated (UR) or downregulate d (DR) in aggressive cancer
1	175412	REPS2	0.14613724	-7.5068116	Cancer UR
2	33909	LOC285588	3.2824067	-1.8452802	Cancer UR
3	22438	C200RF112	2.62604692	-4.0643154	Cancer DR
4	337363	DIPAS	1.06084697	-3.2177178	Cancer UR
5	165657	TPRX1	1.79654397	-2.8351934	Cancer UR
6	38966	FLJ16369	1.13600879	-2.5978003	Cancer UR
7	44021	LOC652255	3.03708564	-1.7358787	Cancer UR
8	41116	LOC642130	2.03361554	-1.9989049	Cancer UR
9	21343	DPRX	2.98927307	-2.5628675	Cancer UR
10	339666	LOC440225	1.72985119	-2.6607478	Cancer UR
11	91353	HS.434989	1.08668984	-2.5889165	Cancer UR
12	40621	LOC642335	.6973359	-2.2656365	Cancer UR
13	369972	GGTLC1	1.04843521	-3.889128	Cancer UR
14	32275	LOC642966	4.72677994	-2.0890642	Cancer UR
15	22944	NKX6-1	2.1845754	-2.4379099	Cancer UR
16	17458	GPC5	0.40241623	-4.2203747	Cancer UR
17	13240	RASGEF1C	0.83201981	-3.3957263	Cancer UR
18	22752	ACOT11	2.83207957	-2.2946388	Cancer UR

Example 4: Identification of Signature 4 Z (comparative example)

A microarray was used to search for a signature of normal vs. aggressive cancer in subtraction-normalized expression data collected from blood samples processed 4 hours after collection. Based on this assay, the 10 genes of Signature 4 were selected and cross validated as described in Example 5, below. Signature 4 had a sensitivity (probability of correctly detecting aggressive cancer) of 64%, and a specificity (probability of correctly identifying normal patients) of 58%.

Table 4: Signature 4

Signature 4 marker #	Illumina Transcript ID	Gene name	Aggressive cancer mean	Normal mean	Upregulated (UR) or downregulate d (DR) in aggressive cancer
1	8276	C30RF26	7.1783549	-54.138047	Cancer UR
2	22171	FOXA3	3.2824067	-1.8452802	Cancer UR
3	1150	FANCB	4.67348819	-1.6783285	Cancer UR
4	162004	ALDH9A1	4.70953157	-1.8584848	Cancer UR
5	334559	LOCI 00133	4.69638401	-1.994684	Cancer UR
6	19987	NR2E1	3.75650279	-2.8100791	Cancer UR
7	90347	HS.413397	2.45548587	-2.5067911	Cancer UR
8	7726	NSUN7	2.1845754	-2.4379099	Cancer UR
9	34808	LOC651044	2.37077671	-2.1346992	Cancer UR
10	364878	PCBP2	3.6810006	-2.3531877	Cancer UR

Example 5: Statistical analysis of microarray data

Working with microarray data can be challenging because large numbers of genes can increase the likelihood of false positives, while a small number of samples can lead to overfitting. These issues can be overcome by using statistical methods to reduce the false rate of positives and using independent training and test data sets (e.g., cross-validation) to avoid overfitting. In particular, instead of using a "typical" 5% significance level, the false discovery rate (FOR) can be controlled to ensure that only 5% of the genes that are discovered are false positives, and Empirical Bayesian estimates can be used to improve test statistics.
Because an overfit model will perform poorly on an independent test set, a good test of the fit of a model is how well is performs on an independent test set. For small sample sizes, splitting data into test and training sets may leave too small of a data set for good training. This issue can be solved by using cross-validation, which splits the data into K-folds, trains the method on K-1 of the folds, and tests the method on the last fold. The ideal split for cross-validation is 10-fold for accurate and precise estimates of diagnostic accuracy. In a 10-fold cross validation, however, there are more than 10 splits because there are many choices for which data points go into the folds. For example, with the microarray data collected as described above, there are 50,979,600 ways to form 90% training/10% testing data sets.
The Empirical Bayesian method was used as follows:

1. The differential gene expression (DE) of macrophages vs. T cells was calculated for each gene. DE is expressed as the log of the ratio of phagocyte to T cell expression: DE = log(GE_P/GE_TC), where GE_P is phagocyte gene expression and GE_TC is T cell gene expression.
2. The mean DE was compared in cancer and control patients with a two- sample t-test. Empirical Bayes estimates of the test statistics "shrink" these toward zero.
3. Calculate a diagnostic signature with K genes: $S = \sum_{i = 1}^{K} w_{i} ({DE}_{i} - μ_{i})$
If S>O, then the patient was diagnosed with cancer.
4. The number of genes K to include in the signature was determined by comparing misclassification rates in independent test sets with cross-validation.

Using the above methods, the markers associated with aggressive prostate cancer in macrophages vs. T cells (Tables 1 and 2) were identified.

Claims

A method for diagnosing or aiding in the diagnosis of prostate cancer in a subject, or for assessing the risk of developing prostate cancer in a subject, or for prognosing or aiding in the prognosis of prostate cancer in a subject, the method comprising the steps of:
(a) measuring the levels of ten or more markers selected from the group of markers identified in Table 1 as Signature 1 markers in a population of the subject's macrophage cells or neutrophil cells;

(b) measuring the levels of the ten or more selected Signature 1 markers in a population of the subject's non-phagocytic cells; and

(c) identifying a difference between the measured levels of the ten or more selected Signature 1 markers in steps (a) and (b),
wherein the identified difference indicates that the subject has said prostate cancer, or indicates that the subject has a risk of developing said prostate cancer, or is indicative of the prognosis of said prostate cancer in the subject, respectively.
A method for assessing the efficacy of a treatment for prostate cancer in a subject, or for monitoring the progression or regression of a prostate cancer in a subject, or for identifying a compound capable of ameliorating or treating prostate cancer in a subject, comprising:
(a) measuring the levels of ten or more markers selected from the group of markers identified in Table 1 as Signature 1 markers in a population of the subject's macrophage cells or neutrophil cells before the treatment, or at a first time point, or before administering the compound to the subject, respectively;

(b) measuring the levels of the ten or more selected Signature 1 markers in a population of the subject's non-phagocytic cells before the treatment, or at the first time point, or before administering the compound to the subject, respectively;

(c) identifying a first difference between the measured levels of the ten or more selected Signature 1 markers in steps (a) and (b);

(d) measuring the levels of the ten or more selected Signature 1 markers in a population of the subject's macrophage cells or neutrophil cells after the treatment, or at a second time point, or after the administration of the compound, respectively;

(e) measuring the levels of the ten or more selected Signature 1 markers in a population of the subject's non-phagocytic cells after the treatment, or at a second time point, or after the administration of the compound, respectively;

(f) identifying a second difference between the measured levels of the ten or more selected Signature 1 markers in steps (d) and (e); and

(g) identifying a difference between the first difference and the second difference, wherein the difference identified in (g) is indicative of the efficacy of the treatment for said prostate cancer in the subject, or is indicative of the progression or regression of said prostate cancer in the subject, or indicates that the compound is capable of ameliorating or treating said prostate cancer in the subject, respectively.
The method of claim 1 or 2, further comprising measuring at least one standard parameter associated with said prostate cancer.
The method of claim 3, wherein the standard parameter is selected from the group consisting of tumor stage, tumor grade, tumor size, tumor visual characteristics, tumor growth, tumor thickness, tumor progression, tumor metastasis tumor distribution within the body, odor, molecular pathology, genomics, or tumor angiograms.
The method of claim 1 or 2, wherein the measured levels are gene expression levels.